Prognostic value of Dicer expression in human breast cancers and association with the mesenchymal phenotype

Abstract

Background: Dicer, a ribonuclease, is the key enzyme required for the biogenesis of microRNAs and small interfering RNAs and is essential for both mammalian development and cell differentiation. Recent evidence indicates that Dicer may also be involved in tumourigenesis. However, no studies have examined the clinical significance of Dicer at both the RNA and the protein levels in breast cancer.

Methods: In this study, the biological and prognostic value of Dicer expression was assessed in breast cancer cell lines, breast cancer progression cellular models, and in two well-characterised sets of breast carcinoma samples obtained from patients with long-term follow-up using tissue microarrays and quantitative reverse transcription-PCR.

Results: We have found that Dicer protein expression is significantly associated with hormone receptor status and cancer subtype in breast tumours (ER P=0.008; PR P=0.019; cancer subtype P=0.023, luminal A P=0.0174). Dicer mRNA expression appeared to have an independent prognostic impact in metastatic disease (hazard ratio=3.36, P=0.0032). In the breast cancer cell lines, lower Dicer expression was found in cells harbouring a mesenchymal phenotype and in metastatic bone derivatives of a breast cancer cell line. These findings suggest that the downregulation of Dicer expression may be related to the metastatic spread of tumours.

Conclusion: Assessment of Dicer expression may facilitate prediction of distant metastases for patients suffering from breast cancer.

Figure 1

Schematic representation of five Dicer mRNA variants and their corresponding proteins according to AceView (http://www.ncbi.nlm.nih.gov/ieb/research/acembly/), transcription from the Dicer gene produces 14 mRNAs, 11 alternatively spliced variants and 3 full-length forms. The three full-length mRNA variants a, b, and c, the corresponding full-length protein (218 kDa) and the two alternatively spliced variants, d and e, and their corresponding proteins (113 and 93 kDa, respectively) are shown. Coloured areas represent the coding region and black bars represent 5′UTR and 3′UTR. Domain structures are represented on proteins.

Figure 2

Expression of Dicer and EMT markers in a human mammary cancer progression cellular model. Expression of Dicer in the human breast tumour progression cellular model: (A) western blot analysis of Dicer expression in the four human breast cell lines, namely HMEC, HMEC+hTERT, HMEC+LT+hTERT, and HMLER (expressing H-rasV12) cells. The signal intensity of Dicer was normalised to that of actin. (B)The relative levels of Dicer mRNA were measured by real-time RT–PCR, and each bar represents the mean±s.d. of the PCRs in triplicate (▪). Dicer protein levels were quantified using Quantity One software (BioRad, Marnes-la-Coquette, France) and expressed as protein relative quantity. The ratios of Dicer/actin of three independent studies were expressed as mean±s.d. (). Morphological characteristics and expression of epithelial and mesenchymal markers in the human breast tumour progression cellular model: (C) Morphological characteristics of the HMEC+hTERT, HMEC+LT+hTERT, and HMLER cells in vitro as assessed by phase-contrast microscopy. Images are shown at × 40 magnification. (D) Western blot analysis of E-cadherin and vimentin expression in the four human breast cell lines, HMEC, HMEC+hTERT, HMEC+LT+hTERT, and HMLER. Actin served as a loading control. E, epithelial phenotype; M, mesenchymal phenotype.

Figure 3

Expression of Dicer in metastasis progression cellular model and in bone metastatic cells. Expression of Dicer in the mouse metastasis progression cellular model: (A) western blot analysis of Dicer expression in the five mouse mammary cell lines, namely 67NR, 168FARN, 4TO7, 66c14, and 4T1. The signal intensity of Dicer was normalised to that of actin. (B) The relative levels of Dicer mRNA were measured by real-time RT–PCR, and each bar represents the mean±s.d. of the PCRs in triplicate (▪). Dicer protein levels were quantified using Quantity One software (BioRad, Marnes-la-Coquette, France) and expressed as protein relative quantity. The ratios of Dicer/actin of three independent studies were expressed as mean±s.d. (). E, epithelial phenotype; M mesenchymal phenotype; E/M, mixed phenotype. Expression of Dicer in two bone metastatic derivatives of MDA-MB-231 cells: (C) western blot analysis of Dicer expression in the three cell lines, namely MDA-MB-231, BO2, and SCP2. The signal intensity of Dicer was normalised to that of actin. (D) The relative levels of Dicer mRNA were measured by real-time RT–PCR, and each bar represents the mean±s.d. of the PCRs in triplicate (▪). Dicer protein levels were quantified using Quantity One software (BioRad, Marnes-la-Coquette, France) and expressed as protein relative quantity. The ratios of Dicer/actin of three independent studies were expressed as mean±s.d. ().

Figure 4

Immunohistochemical analysis of Dicer protein expression in normal and breast cancer tissues. (A) Low-intensity staining in the cytoplasm of epithelial luminal normal breast cells. Non-specific staining of mast cells. (B) Low-intensity staining in the cytoplasm of infiltrating ductal carcinomatous cells. (C) High-intensity staining in the cytoplasm of infiltrating ductal carcinomatous cells. Images are shown at magnification, × 40 (panels A–C).

Figure 5

Kaplan–Meier estimates of metastatic-free survival by Dicer's expression among the mRNA population. >8 cDNA relative quantity (7 out of 43 progressions; 8-year event-free survival probability 88.3% (95% CI: 77.3–99.3); ⩽8 cDNA relative quantity (22 out of 45 progressions; 8-year event-free survival probability 49.1% (95% CI: 33.6–64.6); log-rank test: P=0.0032.